1a.Objectives (from AD-416):
The main problems in yellow perch culture are the lack of genetically defined broodstock with enhanced traits for year-round production, poor larval survival, slow growth, and disease susceptibility. These problems are being pursued via a long-term genetic selection program to produce superior germplasm and complementary studies to understand the physiological basis for performance traits of interest. This project aims to integrate genetic, molecular, physiological, and nutritional approaches to develop superior pathogen-free broodstocks and improved production methods for commercial industry. We will focus on the following objectives:
Objective 1: Develop yellow perch broodstock, define growth and viral hemorrhagic septicemia (VHS)-resistant phenotypes, characterize genetic diversity, and evaluate genotype x environment interaction for growth.
Objective 2: Characterize critical pathways involved in growth and VHS resistance in yellow perch through gene expression and physiological studies.
Objective 3: Improve early survival and methods for producing feed-trained fingerlings.
Objective 4: Develop and evaluate challenge assays, detection tools, and vaccines for protecting yellow perch and other Great Lakes region species of fish from VHS.

1b.Approach (from AD-416):
For objective 1, we will characterize important phenotypes and genotypes in yellow perch broodstocks. Third-generation progeny will undergo performance testing for improved growth and survival and decreased susceptibility to the viral hemorrhagic septicemia (VHS) virus. This will involve evaluation of genotype x environment interactions in laboratory and industrial settings. This information will provide estimates for the heritability of desired traits (growth and VHS resistance) and a better understanding of the sources of phenotypic variation for these traits.
For objective 2, we will generate genomic resources to aid in development of molecular tools to genotype and quantify expression of genes involved with growth and immunity in yellow perch. We will also develop proteomic tools that will enable us to measure and characterize the function of critical proteins (hormones and immune markers) important to growth and immunity for this species. Genomic tools will come from next-generation sequencing efforts to characterize the transcriptomes (expressed genes) of key tissues involved with growth and immunity. Proteomic tools will be developed to characterize biochemical pathways that underlie growth and immunity in yellow perch. Lastly, in vitro methods will be used to characterize how viral proteins impact cellular antiviral recognition and response pathways that impact how yellow perch combat VHS infection, and how the virus might evade or suppress immune pathways in this species.
For objective 3, we will evaluate and test use of larval specialty micro-diets (SMD) as substitutes for live-prey diets to improve larval survival and standardize and reduce overall costs of producing high-quality yellow perch fingerlings. We will evaluate performance measures (first-feeding, swim bladder inflation, development, survival and growth) of genetically defined larval perch broodstock progeny that are reared under either a control live-diet regimen (typical for industry) or a dietary regimen where live prey is progressively substituted with SMD.
For objective 4, we will utilize a standardized VHS challenge model to characterize the disease process and susceptibility of perch broodstocks to VHS infection. We will also develop new diagnostic tools to detect VHS and use these detection tools to evaluate how vaccines and vaccination strategies increase protective immunity against VHS infection. For the challenge assay, genetically defined perch will be exposed to varying doses of VHS virus, and survivors will be re-infected with VHS to characterize resistance and protective immunity to this pathogen. To detect this pathogen, we will develop and validate a novel polymerase chain reaction (PCR) assay that reliably speeds up VHS detection in a cost-effective manner. For vaccination and vaccination strategies, we will characterize protective immunity in perch, evaluating the efficacy and duration of existing and new vaccines for VHS and how new and existing adjuvants extend the efficacy of these vaccines.

3.Progress Report:
Progress was made on all four objectives. We continued to produce and evaluate improved yellow perch broodstocks. Performance trials of the third generation (F3) progeny were completed, and the highest performing individuals were selected, tagged, and are being cycled to enable reproduction in 2013. Genotype by Environment (GxE) studies were completed at two commercial facilities, and data on growth and survival and genetic material were collected at these sites. Cross by strain tests for susceptibility of F3 progeny to the viral hemorrhagic septicemia virus (VHSv) have shown strain-specific differences in survival to pathogen exposure. A non-lethal method for identifying gender in yellow perch was developed. This method was transferred to a major yellow perch producer, enabling this producer to cost-effectively sort sexes for broodstock management and development. We continued to characterize critical pathways that underlie growth, immunity, and susceptibility to the VHSv pathogen, and recently demonstrated that the immune response differs between male (higher) and female (lower) yellow perch following exposure to a pathogen mimetic. Given the higher growth and apparent resistance to pathogen challenge in female perch, development of all-female broodstocks may improve production of perch in commercial aquaculture. Research is underway to develop and test diets that sustain high growth and immunity in genetically-improved yellow perch; this research will address suspected nutritional deficiencies that cause skeletal abnormalities and disease in rapidly growing perch. Research is demonstrating that various structural genes of the VHSv contribute to its virulence in fish cell-lines, whereas another specific gene stimulates the innate immune response in fish cell-lines. The stimulating effect of this VHSv structural gene on the innate immune system suggests it can be used as an adjuvant to boost immune performance in vaccines. We continued to develop and test methods that improve early survival and feed training of yellow perch fingerlings. Improvements to early feeding and rearing resulted in higher egg survival from each egg strand, meaning that reproductive success was improved. Findings are also enabling refinements to live feeds that will further improve survival of larval yellow perch. Development of a rapid test for detecting VHSv in yellow perch was completed. The standardized reverse transcriptase polymerase chain reaction (StaRT-PCR) shows high specificity to the VHSv and displays higher dynamic range in quantifying virus levels and lower false positive and false negative events than present assays. The progress resulting from this research should impact efforts to select yellow perch broodstocks with high growth and low susceptibility to the VHS virus, improve reproductive performance and fingerling quality for yellow perch producers, facilitate the development and testing of vaccines that minimize the effects of the VHS virus, and enable cost-effective surveillance and detection of the VHS virus in agricultural and fisheries production settings.

4.Accomplishments
1.
Reproductive efficiency of yellow perch broodstock is improved. As part of an ongoing research effort to increase yellow perch fingerling production cost-effectively, ARS collaborators at the University of Wisconsin-Milwaukee repeated an early life stage husbandry trial in January, 2012, using an egg ribbon density of nine egg strands/tank. The diets for first-feeding sac-fry through the fingerling stage were consistent with trials previously conducted. Fingerling production was 57,500 per tank, and survival was 28%, which represents a 40% increase over previous trials. Based on the results from these trials and previous trials, optimal yellow perch fingerling production is achieved with nine egg strands per flow-through tank. Confirmation of this finding results in a direct increase in reproductive efficiency in yellow perch, thusly increasing availability of perch fingerlings for the aquaculture industry.

2.
A non-lethal and rapid method for reliable identification of gender in yellow perch has been developed. On average, yellow perch females grow faster than males, so aquaculture producers would like to separate yellow perch by gender. Despite this difference in size, it is difficult to distinguish the sex of immature animals without causing harm or death to the animal. To address this, ARS researchers at Milwaukee, Wisconsin have developed criteria that enable gender identification in yellow perch, based upon the coloring and shape of the external reproductive openings, with an accuracy exceeding 97%. This non-lethal method provides a useful and practical tool that will enable aquaculture producers and researchers to sort sexes for many uses, including:.1)development and management of broodstocks (breeding stock) prior to, and during, breeding;.2)conducting multi-tank replicate experiments aimed at studying gender-specific differences in yellow perch; and.3)identification of the fastest growing individuals of each sex in yellow perch broodstock genetic-selection programs dedicated to developing improved growth performance in this species. The criteria developed for gender identification in yellow perch are easily applied with minimal training and do not require costly laboratory procedures. This cost-effective method will enable producers to develop and manage yellow perch broodstocks in a manner that reduces stress to the animals and minimizes overall animal losses, which increases production efficiency in aquaculture operations.

3.
Third generation of genetically-improved yellow perch reach market size four months sooner. Select high performers of yellow perch strains were identified, tagged, and their genetic material was collected for genotyping by ARS researchers at Milwaukee, Wisconsin and their University of Wisconsin at Milwaukee collaborators. As a result of two generations of selection, the time needed to reach market size has been reduced from eleven to seven months. Performance trials of progeny in commercial production systems were conducted. Data on growth and survival have been obtained, and genetic material has been collected from harvested progeny grown in these commercial settings. A cross x strain comparison of survival of yellow perch progeny exposed to the viral hemorrhagic septicemia virus (VHSv), using a standardized disease challenge study, shows that there are strain-specific differences in survival between progeny. These efforts have enabled a better understanding of genetic differences in susceptibility of yellow perch to this pathogen, and they will enable further genetic improvement of commercially-important traits (growth and disease susceptibility) that increase productivity of yellow perch farming. Faster growth to market size will enable producers to have multiple production cycles in a single year, which increases profitability of commercial yellow perch aquaculture operations.

4.
A rapid test for detecting the viral hemorrhagic septicemia virus (VHSv) in aquaculture species has been developed. This virus has spread into the Great Lakes region, caused fish die-offs and potentially threatens the aquaculture industry. ARS researchers at Milwaukee, Wisconsin, along with collaborators at the University of Toledo and University of Wisconsin, have developed a standardized reverse transcriptase polymerase chain reaction (StaRT-PCR) test to quantify the VHSv pathogen. The StaRT-PCR test is specific to the VHSv pathogen; is rapid; and displays a higher detection range and accuracy than existing tests. The higher accuracy of the StaRT-PCR assay results in significantly lower false positive and false negative rates seen with existing methods. Given the high accuracy and reduced detection time, the StaRT-PCR assay can accelerate testing for susceptibility of perch strains to VHSv; facilitate the development and evaluation of vaccines for this pathogen; and reduce the time and costs of VHSv surveillance to aquaculture producers.

5.
Specific genes of the viral hemorrhagic septicemia virus (VHSv) affect the fish immune system in different ways. ARS collaborators at the University of Toledo have found that two specific proteins of the VHSv pathogen negatively affect the immune response in fish, which enables the VHSv pathogen to infect and replicate within host fish cells. In contrast, other findings suggest that another specific protein of the VHSv gene stimulates the immune response, suggesting its use as an adjuvant to enhance the cellular immune response to vaccines. Knowledge of how particular VHSv proteins affect the viral recognition and response pathway in fish will enable development of more targeted and effective methods for combating this pathogen in important aquaculture species.

6.
Gender-specific differences in fish immunity identified in yellow perch. Based on previous studies in yellow perch, ARS researchers at Milwaukee, Wisconsin questioned whether the expression pattern of genes involved in the growth, metabolism, and immunity in male and female yellow perch function differently. ARS scientists hypothesized that the expression of specific genes would be different in male and female yellow perch that were treated with lipopolysaccharide, a compound that mimics a pathogen. The lipopolysaccharide did turn on certain genes in yellow perch, but the response was generally stronger in males. Findings suggest that these specific genes are important to immune function in yellow perch, and that sex-specific differences in expression occur in finfish like perch. A better understanding of how stress, gender and hormones (sex steroids) interact to influence immune responses in finfish could provide mechanistic information that will improve the understanding, management, and treatment of common stressors that affect the disease susceptibility of finfish in the aquaculture industry.